The invention relates to biological methods of assaying analytes.
Biological assays for analyte detection generally involve attaching to the analyte (e.g., nucleic acids, proteins, hormones, lipids, or cells) a signal-generating moiety. Fluorescence-based bioassays require the detection of weak fluorescence signals. In a typical assay, the analyte is deposited onto a solid substrate such as a microscope slide or a glass chip. After undergoing biochemical treatment and fluorescent staining, the slide is examined with an optical instrument such as a fluorescence microscope. Light of certain wavelengths is applied to the slide, and the fluorescent emission from the deposited biomaterial is collected as a signal.
Transparent soda-lime and borosilicate glasses are commonly used as substrates to support fluorescently labeled samples. However, many of these materials exhibit significant autofluorescence, have finite absorbance, and can produce fluorescent emission throughout the visible region. A typical soda-lime glass slide can produce background fluorescence equivalent to a layer of a commonly used fluorescent dye with a surface density of more than 1xc3x97109 fluorophors/cm2. This background fluorescence along with noise from other sources, such as stray light, such as stray light and Rayleigh and Raman scattering, can obscure the detection of weak fluorescent signals from the analyte, limiting the sensitivity of the assay.
Further, in many fluorescent assays for nucleic acid detection, nucleic acids are attached to a solid support via chemical linkers. Such linkers often are autofluorescent and can introduce background fluorescence.
The invention features improvements in biological assays. In one aspect, the invention features fluorescence-based assays that have a significantly reduced signal background compared to conventional assays. These assays include the steps of: (i) providing an opaque glass support with a surface that is in contact with a sample containing an analyte (e.g., a protein, a nucleic acid, a polysaccharide, a lipid, or a cell), where the analyte, in present in the sample, is labeled with a fluorochrome; (ii) illuminating the surface with light at a wavelength that excites the fluorochrome; and (iii) detecting fluorescent emission from the surface as an indication for the presence of the compound in the sample. As used herein, xe2x80x9can opaque glass supportxe2x80x9d refers to a glass support that is impervious to the excitation and emission lights of the fluorochrome used in an assay.
In the above assays, a reflective surface can be used in lieu of an opaque glass support. By xe2x80x9ca reflective surfacexe2x80x9d is meant that, when incoming light is directed to the surface perpendicularly, the surface reflects at least about 15% (e.g., at least 25%, 50%, 75%, or 90%) of the incoming light, while transmitting no more than 20% (e.g., no more than 10%, 5%, or 1%) of the light. In assays using a reflective surface, the excitation light can be directed to the surface at an angle, i.e., non-perpendicularly. A reflective surface can be, for instance, metallic (e.g., chromium or aluminum) surface or metalloid (e.g., silicon) surface.
In the fluorescence assays of the invention, the analyte can be bound to the surface via a capture probe that binds specifically to the analyte and is immobilized on the surface.
In another aspect, the invention features methods of efficiently attaching nucleic acid to a metallic or metalloid surface. These methods include the steps of: (i) providing a solution that contains the desired nucleic acid; (ii) denaturing the nucleic acid in the solution; (iii) applying the solution to the metallic (e.g., chromium or aluminum) or metalloid (e.g., silicon) surface; and (iv) allowing the solution to dry on the surface, thereby attaching the nucleic acid to the surface. In these methods, the nucleic acid can be denatured in an alkaline solution (e.g., a NaOH solution that has a pH of at least about 11, or by being heated to a temperature and for a time sufficient to denature the nucleic acid. A microscopy mounting medium, e.g., Gel/Mount(trademark) (Biomeda Corp., Foster City, Calif.), can be optionally applied to the metal or metalloid surface to enhance the attachment of the nucleic acid to the surface.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications and any other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.